PD-L1 targeting compound, radioactive tracer agent and preparation method and application of PD-L1 targeting compound and radioactive tracer agent

Abstract

The invention is suitable for the technical field of biological medicine and nuclear medicine, and provides a PD-L1 targeting compound, a radioactive tracer agent and a preparation method and application of the PD-L1 targeting compound and the radioactive tracer agent. The radioactive tracer provided by the invention has strong binding force with PD-L1, can accurately locate PD-L1 in vivo, has excellent in-vivo targeting performance, and can achieve the purpose of tumor molecular imaging through nuclear medicine imaging; meanwhile, noninvasive visualization of PD-L1 expression is achieved, non-invasive tumor diagnosis can be effectively completed through small animal PET / CT verification, and the clinical application prospect is wide; in addition, the PD-L1 targeting compound and the radioactive tracer derived from the PD-L1 targeting compound further have the advantages of being simple in preparation process, low in cost, high in specificity, good in in-vivo and in-vitro stability, long in imaging period, easy to clinically convert and the like, and the comprehensive performance is outstanding.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine and nuclear medicine technology, and particularly relates to a compound targeting PD-L1, a radioactive tracer and a preparation method and application thereof. BACKGROUND

[0002] Programmed Death-Ligand 1 (PD-L1) is a key molecule mediating tumor immune escape. Its abnormally high expression inhibits T cell function by binding to PD-1 on the surface of T cells, thereby promoting the occurrence and development of tumors. At present, PD-1 / PD-L1 inhibitors have been widely used in clinical practice, but some patients develop drug resistance due to tumor microenvironment regulation or alternative pathway activation, affecting the treatment effect.

[0003] The existing PD-L1 detection methods have obvious limitations: image technology examination (such as X-ray, CT, MRI, and ultrasound) evaluates the efficacy by morphological changes of the tumor, and significant changes in tumor volume after chemotherapy usually lag for more than two weeks, making it difficult to reflect the treatment response in a timely manner; tumor marker detection is interfered by many factors such as inflammation, infection, and drugs, and the expression level fluctuates greatly and individual differences are significant, limiting the accuracy; molecular biology detection (including fluorescence in situ hybridization, reverse transcription polymerase chain reaction, high-throughput sequencing, and immunohistochemistry) requires invasive sampling of patients, and PD-L1 expression in different parts of the tumor tissue is heterogeneous, so local specimens cannot fully reflect the overall condition of the disease, and the detection standards are not uniform, limiting its application as a predictive biomarker.

[0004] With the development of medical imaging technology, nuclear medicine PET / CT imaging technology based on molecular recognition and tracing provides a new direction for tumor treatment efficacy evaluation. This technology does not require invasive procedures and can achieve in vivo visualization and tracking of target points in a non-invasive manner, can accurately diagnose lesions to overcome tumor heterogeneity and sampling bias, can reveal early pathological changes after treatment, and can provide guidance for individualized treatment plans, and can evaluate the dynamic changes of pathophysiology in vivo without the need for ex vivo detection. Based on these advantages, the use of nuclear medicine PET / CT imaging technology to achieve non-invasive monitoring of PD-L1 activity has important value for evaluating the efficacy of PD-L1 targeted therapy and accurately determining the effect of anti-tumor drugs in a timely manner. SUMMARY

[0005] Therefore, the present application provides a compound targeting PD-L1 and a radioactive tracer derived therefrom, aiming to solve the problems of existing PD-L1 detection methods such as lag, invasiveness, difficulty in reflecting dynamic changes, and incomplete evaluation.

[0006] A first aspect of the embodiments of the present application provides a compound targeting PD-L1, the compound targeting PD-L1 has a structure shown in the following formula (I) or formula (II) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a hydrate thereof: Formula (I): ; Formula (II): .

[0007] A second aspect of the embodiments of the present application provides an intermediate of the compound targeting PD-L1 in the first aspect, the intermediate of the compound shown in the formula (II) is shown in the following formula (IV): Formula (IV): ; In the formula (IV), the structure of -NHBoc is

[0008] A third aspect of the embodiments of the present application provides a preparation method of the compound targeting PD-L1 in the first aspect, comprising: After removing Boc protection on the compound shown in the following formula (III), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out condensation reaction, to obtain the compound targeting PD-L1 shown in the formula (I); After removing Boc protection on the compound shown in the following formula (IV), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out condensation reaction, to obtain the compound targeting PD-L1 shown in the formula (II); Formula (III): ; Formula (IV): .

[0009] A fourth aspect of the embodiments of the present application provides a radioactive tracer, comprising the compound targeting PD-L1 in the first aspect or the compound targeting PD-L1 prepared by the preparation method in the third aspect and a radionuclide.

[0010] A fifth aspect of the embodiments of the present application provides a preparation method of the radioactive tracer in the fourth aspect, comprising: Mixing reaction of the radionuclide and the compound targeting PD-L1 is carried out to obtain the radioactive tracer.

[0011] The sixth aspect of the embodiments of the present application provides application of the radioactive tracer in the fourth aspect or the radioactive tracer prepared by the preparation method in the fifth aspect in preparation of a preparation for detecting expression level of PD-L1 in a tumor or in preparation of a preparation for diagnosing a tumor with high expression of PD-L1.

[0012] Compared with the prior art, the embodiments of the present application have the following beneficial effects: The radioactive tracer provided by the embodiments of the present application has strong binding force with PD-L1, can accurately locate PD-L1 in vivo, has excellent in-vivo targeting performance, and can realize the purpose of tumor molecular imaging through nuclear medical imaging; at the same time, the radioactive tracer realizes non-invasive visualization of PD-L1 expression, can effectively complete non-invasive diagnosis of a tumor through small animal PET / CT verification, and has wide clinical application prospects; in addition, the compound targeting PD-L1 and the radioactive tracer derived therefrom also have the advantages of simple preparation process, low cost, high specificity, good in-vivo and in-vitro stability, long imaging cycle, and easy clinical transformation, and have outstanding comprehensive performance. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 A synthesis route of the compound CB01 targeting PD-L1 provided for the embodiment 1 of the present application; Figure 2 A nuclear magnetic hydrogen spectrum of the compound 1 provided for the embodiment 1 of the present application; Figure 3 A high-resolution mass spectrum of the compound 1 provided for the embodiment 1 of the present application; Figure 4 A high-resolution mass spectrum of the compound CB01 targeting PD-L1 provided for the embodiment 1 of the present application; Figure 5 A synthesis route of the compound CB02 targeting PD-L1 provided for the embodiment 2 of the present application; Figure 6 A nuclear magnetic hydrogen spectrum of the intermediate 4 provided for the embodiment 2 of the present application; Figure 7 A high-resolution mass spectrum of the intermediate 4 provided for the embodiment 2 of the present application; Figure 8 A nuclear magnetic hydrogen spectrum of the intermediate 5 provided for the embodiment 2 of the present application; Figure 9 A high-resolution mass spectrum of the intermediate 5 provided for the embodiment 2 of the present application; Figure 10 A high-resolution mass spectrum of the compound CB02 targeting PD-L1 provided for the embodiment 2 of the present application; Figure 11 A radioactive tracer provided for the embodiment 3 of the present application 68Radio-HPLC analysis results of [68Ga]Ga-CB01; Figure 12 Radio-tracer provided for Example 3 of the present application 68 Radio-HPLC analysis results of [68Ga]Ga-CB02; Figure 13 Radio-tracer provided for Example 4 of the present application 68 Results of in vitro stability study of [68Ga]Ga-CB01; Figure 14 Radio-tracer provided for Example 4 of the present application 68 Results of in vitro stability study of [68Ga]Ga-CB02; Figure 15 Radio-tracer provided for Example 6 of the present application 68 Saturation curves of [68Ga]Ga-CB01 and radio-tracer 68 [68Ga]Ga-CB02; Figure 16 Radio-tracer provided for Example 6 of the present application 68 Uptake curves of [68Ga]Ga-CB01 and radio-tracer 68 [68Ga]Ga-CB02; Figure 17 Radio-tracer provided for Example 7 of the present application 68 Blood clearance curves of [68Ga]Ga-CB01 and radio-tracer 68 [68Ga]Ga-CB02; Figure 18 Comparison chart of small animal PET / CT imaging provided for Example 8 of the present application; Figure 19 Radio-tracer provided for Example 9 of the present application 68 Radioactivity distribution histograms of [68Ga]Ga-CB01 and radio-tracer 68 [68Ga]Ga-CB02 in tissues of HCC78 tumor-bearing mice; Figure 20 Target / non-target ratio histogram provided for Example 9 of the present application; Figure 21 Phosphor screen autoradiograph of corresponding tissues after injection of radio-tracer 68 [68Ga]Ga-CB01 provided for Example 10 of the present application; Figure 22 Phosphor screen autoradiograph of corresponding tissues after injection of radio-tracer 68 [68Ga]Ga-CB02 provided for Example 10 of the present application; Figure 23The tumor tissue section immunofluorescence staining results provided for Example 11 of the present application. DETAILED DESCRIPTION

[0014] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0015] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0016] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0017] In order to solve the problems of the existing PD-L1 detection method, such as hysteresis, invasiveness, difficulty in reflecting dynamic changes, and incomplete evaluation, the present application provides a compound targeting PD-L1, which has the structure shown in the following formula (I) or formula (II) or its stereoisomer, tautomer, pharmaceutically acceptable salt, hydrate: Formula (I): ; Formula (II): .

[0018] The pharmaceutically acceptable salt refers to the organic salt and inorganic salt of the compound of the present application. The pharmaceutically acceptable salt is well known to those skilled in the art. The pharmaceutically acceptable salt includes but is not limited to inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, etc., and organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, etc., or salts obtained by other methods such as ion exchange method as recorded in the literature.

[0019] Similarly, besides the pharmaceutically acceptable salts of the compounds, the stereoisomers (such as enantiomers, diastereomers) of the compounds, the tautomers (such as keto-enol tautomers) of the compounds, the hydrates (crystalline water forms) of the compounds, etc. usually have similar biological activities, and can be naturally formed during preparation or storage, and also belong to the technical solutions under the same concept of the first aspect of the present application.

[0020] The present application further provides intermediates of the above-mentioned PD-L1 targeting compounds, the intermediate of the compound represented by formula (I) is represented by formula (III), and the intermediate of the compound represented by formula (II) is represented by formula (IV). Formula (III): ; Formula (IV): .

[0021] In formula (III) and formula (IV), the structure of -NHBoc is (the structure of -NHBoc in specific embodiment 1-2). The intermediate represented by formula (III) can be prepared by the person skilled in the art based on the prior art, such as by the method of specific embodiment 1.

[0022] The present application further provides a preparation method of the above-mentioned PD-L1 targeting compound, which comprises: After removing the Boc protection of the compound represented by formula (III) below, tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropyl ethylamine are added to carry out condensation reaction, to obtain the PD-L1 targeting compound represented by formula (I); After removing the Boc protection of the compound represented by formula (IV) below, tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropyl ethylamine are added to carry out condensation reaction, to obtain the PD-L1 targeting compound represented by formula (II); Formula (III): ; Formula (IV): .

[0023] In one or more embodiments, the method for removing the Boc protecting group of the compound represented by formula (III) or formula (IV) can be: dissolving the compound represented by formula (III) or formula (IV) in dichloromethane, adding trifluoroacetic acid to react, to obtain the first intermediate after removing the Boc protection.

[0024] In one or more embodiments, the molar ratio of the compound of formula (III) or formula (IV) to tetraazacyclododecane tetraacetic acid-succinimidyl ester (DOTA-NHS) and N,N-diisopropylethylamine (DIPEA) is (23-27):(35-40):(135-160), preferably 25:38:150.

[0025] In one or more embodiments, the method for preparing the compound of formula (IV) comprises: After removing the Boc protection from the compound of formula (III), the compound of formula (IV) is obtained by condensation reaction of adding tert-butyloxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzenetriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine. After removing the Boc protection from the compound of formula (III), the compound of formula (IV) is obtained by condensation reaction of adding tert-butyloxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzenetriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine.

[0026] The present application further provides a radioactive tracer comprising the PD-L1 targeting compound prepared by the above method or the PD-L1 targeting compound prepared by the above method and a radionuclide.

[0027] In one or more embodiments, the radionuclide is selected from 18 F, 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb 、227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd,157 Gd and 166 one of Dy; preferably 68 Ga.

[0028] Preferably, the structure of the radioactive tracer is shown as formula (V) and formula (VI): Formula (V): ; Formula (VI): .

[0029] The application further provides a preparation method of the radioactive tracer, comprising: Mixing and reacting the radionuclide and the PD-L1 targeting compound to obtain the radioactive tracer.

[0030] In one or more embodiments, the radionuclide is 68 For example, 3.5-10 nmol of the PD-L1 targeting compound is mixed with 200 μL of GaCl3 eluent (74 MBq-100 MBq) to add 200 μL of acetic acid-sodium acetate buffer solution (0.25 M), adjust the pH of the reaction system to 4-4.6, and then uniformly mix at 95°C for 10-15 min. After the reaction is completed, the system is cooled to room temperature to obtain the radioactive tracer. 68 For example, 3.5-10 nmol of the PD-L1 targeting compound is mixed with 200 μL of GaCl3 eluent (74 MBq-100 MBq) to add 200 μL of acetic acid-sodium acetate buffer solution (0.25 M), adjust the pH of the reaction system to 4-4.6, and then uniformly mix at 95°C for 10-15 min. After the reaction is completed, the system is cooled to room temperature to obtain the radioactive tracer.

[0031] The application further provides application of the radioactive tracer or the radioactive tracer prepared by the preparation method in preparation of a preparation for detecting expression level of PD-L1 in a tumor or in preparation of a preparation for diagnosing a tumor with high expression of PD-L1.

[0032] The tumor includes any one of lymphoma, multiple myeloma, and solid tumor, for example, lung cancer, liver cancer, pancreatic cancer, gastric cancer, colon cancer, thyroid cancer, head and neck tumor, etc., but is not limited thereto.

[0033] The PD-L1 targeting compound and the radioactive tracer derived therefrom are described in detail in the following specific examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0034] Cell culture: HCC78 cells are cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-gentamicin mixture (P-S-G); and cultured in a 37 °C, 5% CO2 humidified incubator.

[0035] Establishment of HCC78 tumor-bearing mouse model: HCC78 cells were subcutaneously injected into the right shoulder of NSG mice, 2x10 6 cells per mouse, and the tumor growth was observed daily within two weeks. When the tumor grew to a defined size, the imaging and in vivo distribution experiments were carried out.

[0036] Example 1 Figure 1 The synthesis route of the compound targeting PD-L1 shown in formula (I) was referred to Figure 1 , and the compound CB01 targeting PD-L1 was synthesized.

[0037] Compound 1 (100 mg, 0.25 mmol) was dissolved in DCM (11.8 mL), N-tert-butoxycarbonyl ethylenediamine (78.98 mg, 0.49 mmol), acetic acid (10 mg, 0.17 mmol) were added, and after stirring at room temperature for 4 hours, sodium triacetoxyborohydride (156.98 mg, 0.74 mmol) was added, and stirring was continued at room temperature for 12 hours, and the reaction was monitored by TLC. After the reaction was completed, water was added for quenching, DCM was used for extraction, the organic phases of the combined extracts were dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by a rotary evaporator. The crude product was separated by silica gel column chromatography to obtain intermediate 2 (110 mg, yield 83%).

[0038] The nuclear magnetic hydrogen spectrum of compound 1 is shown in Figure 2 , and the high-resolution mass spectrum is shown in Figure 3 .

[0039] Intermediate 2 (20 mg, 0.04 mmol) was dissolved in DCM (0.3 ml), TFA (25.31 mg, 0.22 mmol) was added, and the reaction was carried out at room temperature for 4 hours, and the reaction progress was monitored by TLC. After the reaction was completed, saturated K2CO3 aqueous solution was added for quenching, DCM was used for extraction, the organic phases of the combined extracts were dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by a rotary evaporator. Then DMF (0.3 mL), DOTA-NHS (17.21 mg, 0.03 mmol), and DIPEA (14.75 mg, 0.11 mmol) were added to the reaction system, and stirring was carried out at room temperature for 8 hours, and the reaction was monitored by HPLC. After the reaction was completed, the reaction system was purified by HPLC (see Table 1), and compound 3 (CB01) (4.7 mg, yield 25%) was obtained after freeze-drying.

[0040] The high-resolution mass spectrum of compound 3 (CB01) is shown in Figure 4 .

[0041] Table 1 HPLC purification method (flow rate: 3 mL / min)

[0042] Example 2 Figure 5 The synthesis route for the compound targeting PD-L1 of formula (II) was synthesized to synthesize the compound CB02 targeting PD-L1.

[0043] Intermediate 2 (300 mg, 0.56 mmol) was dissolved in DCM (6 ml), TFA (383.04 mg, 3.36 mmol) was added, and the reaction was carried out at room temperature for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, saturated K2CO3 aqueous solution was added to quench, extracted with DCM, and the combined organic phases were dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by rotary evaporator. Then it was dissolved in DMF (6 ml) in the reaction system, and N-(tert-butoxycarbonyl)-L-proline (180.92 mg, 0.84 mmol), HATU (319.77 mg, 0.84 mmol) and DIPEA (434.21 mg, 3.37 mmol) were added, and stirred at room temperature for 12 h, and the reaction was monitored by TLC. After the reaction was completed, water was added to quench, extracted with ethyl acetate, and the combined extracts were dried over Na2SO4, filtered, and the solvent was evaporated by rotary evaporator, and column chromatography gave intermediate 4 (156 mg, yield 44%).

[0044] The nuclear magnetic resonance spectrum of hydrogen of intermediate 4 is shown in Figure 6 The high resolution mass spectrum is shown in Figure 7

[0045] Intermediate 4 (100 mg, 0.159 mmol) was dissolved in DCM (2 mL), TFA (108.57 mg, 0.95 mmol) was added, and the reaction was carried out at room temperature for 4 h, and the reaction progress was monitored by TLC. After the reaction was completed, saturated K2CO3 aqueous solution was added to quench, extracted with DCM, and the combined organic phases were dried over anhydrous Na2SO4, filtered, and the solvent was evaporated by rotary evaporator. Then it was dissolved in DMF (6 ml) in the reaction system, and tert-butoxycarbonyl 6-aminohexanoic acid (51.9 mg, 0.24 mmol), HATU (120.61 mg, 0.32 mmol) and DIPEA (81.89 mg, 0.63 mmol) were added, and stirred at room temperature for 2 h, and the reaction was monitored by TLC. After the reaction was completed, water was added to quench, extracted with ethyl acetate, and the combined extracts were dried over Na2SO4, filtered, and the solvent was evaporated by rotary evaporator, and column chromatography gave intermediate 5 (59 mg, yield 29.3%).​

[0046] The nuclear magnetic hydrogen spectrum of intermediate 5 is shown in Figure 8 and the high resolution mass spectrum is shown in Figure 9 .

[0047] Intermediate 5 (15 mg, 0.02 mmol) was dissolved in DCM (0.3 ml), TFA (13.99 mg, 0.12 mmol) was added, and the reaction was allowed to proceed at room temperature for 4 h, with TLC monitoring of the reaction progress. After the reaction was completed, saturated aqueous K2CO3 solution was added to quench, and extraction was performed with DCM, the combined organic phases were dried over anhydrous Na2SO4, filtered, and the solvent was evaporated using a rotary evaporator. Then DOTA-NHS (15.04 mg, 0.03 mmol) and DIPEA (3.87 mg, 0.03 mmol) were added to the reaction system, and stirring was performed at room temperature for 4 h, with HPLC monitoring of the reaction. After the reaction was completed, the reaction system was purified by HPLC, and after lyophilization, compound 6 (CB02) (2.3 mg, yield 11.1%) was obtained.

[0048] The high resolution mass spectrum of compound 6 (CB02) is shown in Figure 10 .

[0049] Example 3 Compound CB01 was eluted from a 4 mL high-purity hydrochloric acid (0.1 M) generator 68 Ge- 68 Ga generator; 68 Compound CB02 was eluted from a 4 mL high-purity hydrochloric acid (0.1 M) generator After 3.5 nmol of compound CB02 was mixed with 200 µL of 68 GaCl3 eluate (74 MBq~100 MBq), 200 µL of acetic acid-sodium acetate buffer solution (0.25 M) was added, the pH of the reaction system was adjusted to 4.6, and then stirring was performed at 95°C for 15 min. After the reaction was completed, the reaction system was cooled to room temperature, and the radiotracer [ 68 Ga]Ga-CB02 was obtained.

[0050] After 3.5 nmol of compound CB02 was mixed with 200 µL of 68 GaCl3 eluate (74 MBq~100 MBq), 200 µL of acetic acid-sodium acetate buffer solution (0.25 M) was added, the pH of the reaction system was adjusted to 4.6, and then stirring was performed at 95°C for 15 min. After the reaction was completed, the reaction system was cooled to room temperature, and the radiotracer [ 68 Ga]Ga-CB02 was obtained.

[0051] The radiotracer [ 68Ga]Ga-CB01 and [ 68 The radiochemical purity (RCP) of Ga]Ga-CB02 and the analytical method of radioactive HPLC are shown in Table 2.

[0052] Table 2. Radioactive HPLC analysis method (flow rate: 1 mL / min)

[0053] [ 68 Ga]Ga-CB01 and [ 68 The radioactive HPLC analysis results of Ga]Ga-CBO2 are as follows: Figure 11 and Figure 12 As shown, from Figure 11 It can be seen from [ 68 The retention time of Ga]Ga-CB01 was 14.67 min, RCP > 99%, and specific activity was 9 ± 0.1 GBq / µmol; from Figure 12 It can be seen from [ 68 The retention time of Ga]Ga-CB02 was 14.82 min, RCP > 99%, and specific activity was 28 ± 0.21 GBq / µmol.

[0054] The formula for calculating specific activity is: Specific activity (GBq / µmol) = Radioactivity (GBq) / Amount of precursor compound (µmol).

[0055] Example 4 Radioactive tracers [ 68 Ga]Ga-CB01 and [ 68 In vitro stability study of Ga-CB02: Stability study of radioactive tracer in PBS: 100 µL of radioactive tracer was mixed with 100 µL of PBS and incubated at 37 °C for 2 h; after incubation, 100 µL of the mixture was taken and the RCP of the radioactive tracer was analyzed by radioactive HPLC.

[0056] In vitro serum stability study of the radiotracer: 100 µL of radiotracer was mixed with 100 µL of human serum and incubated at 37 °C for 2 h. After incubation, 200 µL of anhydrous ethanol was added to the incubation system, and then the incubation system was centrifuged at 12000 rpm for 5 min. The supernatant was collected, filtered, and the RCP of the radiotracer was analyzed by radioactive HPLC.

[0057] Radioactive tracers [ 68 The results of the in vitro stability study of Ga]Ga-CB01 are as follows: Figure 13 As shown, Figure 13(a) PBS stability study, Figure 13 (b) in vitro serum stability study. From Figure 13 It can be seen from the above that the in vitro PBS stability of the radiotracer 68 Ga]Ga-CB01 and the in vitro human serum stability are good, and the RCPs at 1 h are all greater than 99%.

[0058] The in vitro stability study results of the radiotracer 68 Ga]Ga-CB02 are shown in Figure 14 Figure 14 (a) PBS stability study, Figure 14 (b) in vitro serum stability study. From Figure 14 It can be seen from the above that the in vitro PBS stability of the radiotracer 68 Ga]Ga-CB02 and the in vitro human serum stability are good, and the RCPs at 1 h are all greater than 99%.

[0059] Example 5 Hydrophilic-lipophilic study: 5 μL of the radiotracer was added to a mixture of 495 μL of HEPES buffer (pH = 7.4) and 500 μL of n-octanol, mixed vigorously, and centrifuged at 5000 rpm for 5 min; after centrifugation, 400 μL of the upper liquid (n-octanol) and 400 μL of the lower liquid (HEPES buffer solution) were taken from the centrifuge tube and centrifuged at 12000 rpm for 5 min; finally, 100 μL of the upper liquid (n-octanol) and 100 μL of the lower liquid (HEPES buffer solution) were taken from the re-centrifuged system, and the radioactivity counts were measured with a γ counter to calculate the oil-water distribution coefficient D (log D ). The calculation formula is: log D =lg ((γ counts in n-octanol) / (γ counts in HEPES buffer)).

[0060] The oil-water distribution coefficients D (log D ) of the radiotracer 68 Ga]Ga-CB01 and the radiotracer 68 Ga]Ga-CB02 were calculated to be -1.80±0.08 and -1.66±0.06, respectively; both of them have excellent hydrophilicity.

[0061] Example 6 Cell receptor affinity study of the radiotracer: (1) The equilibrium dissociation constant (Kd) was determined by saturation experiment K d ​To evaluate the binding affinity of the radiotracer to PD-L1, a saturation binding assay was performed on HCC78 cells. HCC78 cells (4 × 10⁻⁶) were used. 5 Cells were incubated with different concentrations of radioactive tracer (0.625–60 nM) for 1 h to determine total binding. To determine nonspecific binding, cells were pretreated with an excess of PD-L1 antibody (20 μg) before the experiment. Cells in both the total binding and nonspecific binding groups were washed three times with cold PBS, followed by lysis with 200 μL / well of NaOH solution (1 M). The radioactivity count of cell lysis products was measured using a gamma counter. All experiments were performed in triplicate.

[0062] (2) Cell uptake experiment: HCC78 cells (4×10⁻⁶) were added to the cell uptake test. 5 Cells were seeded in 24-well plates (number per well) and incubated with a radiotracer for 15, 30, 60, 90, and 120 min to assess uptake over time. For blocking experiments, cells were pre-incubated with PD-L1 antibody (20 μg) for 1 hour before adding the radiotracer. After incubation, the culture medium was removed, cells were washed with PBS, and then lysed with 200 μL of 1 M NaOH. Radioactivity was then measured using a gamma counter. All experiments were performed in triplicate.

[0063] Radioactive tracers [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 The saturation curve of Ga-CB02 is as follows: Figure 15 As shown, radioactive tracer [ 68 Ga]Ga-CB01 K d The concentration is 98.96 nM, and the radioactive tracer [ 68 Ga]Ga-CB02 K d It is 51.67 nM; from Figure 15 as well as K d The values ​​show that the radioactive tracer [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 Ga]Ga-CB02 has a good affinity for HCC78 cells. K d The values ​​are all in the nM range.

[0064] Radioactive tracers [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 The uptake curve of Ga-CBO2 is as follows: Figure 16 As shown, from Figure 16As can be seen from this, the radioactive tracer [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 Ga-CB02 exhibited rapid and specific uptake within 15 minutes, gradually increasing over time until stabilizing at 90 minutes; the uptake inhibition by the PD-L1 antibody confirmed the radioactive tracer's […]. 68 Ga]Ga-CB01 and radioactive tracers[ 68 The binding of Ga]Ga-CBO2 to PD-L1 is specific.

[0065] Example 7 A radioactive tracer was injected via the tail vein into normal BALB / c female mice (n=3). 68 Ga]Ga-CB01, [ 68 Ga]Ga-CB02 (7.4 MBq each). Mouse blood was then collected at 1, 3, 5, 10, 15, 30, 45, 60, 90, and 120 min, weighed, and the radioactivity count was measured using a gamma counter. %ID / g was calculated, and the blood clearance half-life of each radiotracer was obtained by fitting blood clearance curves using Drug And Statistics (DAS) software. The formula for calculating %ID / g is: %ID / g = ((measured radioactivity count) / (total injected radioactivity count)) / mass of tissue (including blood).

[0066] Radioactive tracers [ 68 Ga]Ga-CB01 and radioactive tracers[ 68 The blood clearance curve of Ga]Ga-CB02 is as follows Figure 17 As shown, from Figure 17 As can be seen, the pharmacokinetic curves of each radiotracer in normal mice conform to a two-compartment model. 68 The phase half-life (t) of Ga]Ga-CB01 1 / 2α The elimination phase half-life (t) is 0.55 minutes. 1 / 2β The time to blood metabolism is 8.878 minutes, which is moderate. Radioactive tracer [ 68 The phase half-life (t) of Ga]Ga-CB02 1 / 2α The elimination phase half-life (t) is 0.646 minutes. 1 / 2β The blood metabolism time is 11.273 minutes, which is moderate.

[0067] Example 8 Small animal PET / CT imaging: HCC78 tumor-bearing mice were randomly divided into an experimental group (n=3) and a blocking group (n=3), and then injected with a radioactive tracer.68 Ga]Ga-CB01 or radiotracer 68 Ga]Ga-CB02, small animal PET / CT imaging was performed. In the experimental group, each tumor-bearing mouse was injected with 200 μL of radiotracer (3.7 MBq) via the tail vein under 2% isoflurane in oxygen. In the blocking group, the tumor-bearing mouse was first injected with PD-L1 antibody (50 μg) via the tail vein, and 24 h later, the radiotracer was injected via the tail vein, and PET / CT imaging was performed. After imaging, the PET / CT images were reconstructed using Nucline NanScan 3.00 software, and image analysis was performed using InterView FUSION 3.0 software.

[0068] The results are shown in Figure 18 , the experimental group had obvious 68 Ga]Ga-CB01 and 68 Ga]Ga-CB02 radioactivity uptake in the tumor site, and the radioactivity accumulation in the inhibition group was significantly reduced; indicating that the radiotracer specifically targets PD-L1 protein and can be used for imaging, efficacy monitoring, etc. of PD-L1 protein-expressing tumors.

[0069] Example 9 Biodistribution: Under gaseous anesthesia (2% isoflurane in oxygen), HCC78 tumor-bearing mice (n=3) were injected with 100 μL of radiotracer 68 Ga]Ga-CB01 or radiotracer 68 Ga]Ga-CB02 (3.7 MBq) via the tail vein. One hour after injection, the HCC78 tumor-bearing mice were subjected to ocular blood sampling, and after sacrifice, important tissues / organs including tumor, muscle, bone, kidney, spleen, liver, intestine, heart, lung, and stomach were collected. The collected blood and tissues were weighed, and then the radioactivity counts were measured by a gamma counter. By comparing the tissue counts with the calibrated counts of the injected radiotracer, the %ID / g and target / non-target (T / NT) ratios were obtained.

[0070] The results are shown in Figure 19 , Figure 20 , Table 3, and Table 4, 60 minutes after injection of radiotracer 68 Ga]Ga-CB01 and 68 Ga]Ga-CB02 in the HCC78 tumor-bearing mouse model, the tumor uptake was 0.38 ± 0.03 %ID / g and 0.47 ± 0.29 %ID / g, respectively, indicating that the radiotracer has tumor targeting properties in PD-L1 positive tumors.

[0071] Table 3 Radiotracer 68Ga]Ga-CB01, 68 Ga]Ga-CB02 biodistribution study

[0072] Table 4 Radiotracers 68 Ga]Ga-CB01, 68 Ga]Ga-CB02 TNT ratio

[0073] Example 10 Phosphor screen autoradiography: HCC78 tumor-bearing mice (n=3) were injected with 100 μL of radiotracer 68 Ga]Ga-CB01 or radiotracer 68 Ga]Ga-CB02 (3.7 MBq) via tail vein under gaseous anesthesia (oxygen containing 2% isoflurane). One hour post-injection, HCC78 tumor-bearing mice were subjected to ophthalmic blood collection and sacrificed. Tumors, muscle, and kidney were collected. Tumors, muscle, and kidney were placed on a phosphor screen and exposed in the dark for 10 min. The phosphor screen was then scanned using a phosphor screen imaging system to obtain images.

[0074] Results are shown in Figure 21 and 22 Phosphor screen analysis results were highly consistent with PET / CT imaging and biodistribution results, further confirming the specific distribution pattern of radiotracer 68 Ga]Ga-CB01 and radiotracer 68 Ga]Ga-CB02 in vivo.

[0075] Example 11 Immunohistochemical staining to verify PD-L1 expression in HCC78 tumor-bearing mouse model: Ex vivo tumor tissues from tumor-bearing mice were fixed in 4% paraformaldehyde for 24 h, then embedded to make paraffin sections; paraffin sections were placed in an oven at 60°C for 30 min, then sequentially immersed in xylene I and xylene II for 10 min each; the sections were sequentially placed in ethanol from high to low for 2 min each (ethanol gradient: 100%, 95%, 90%, 80%, 70%); then the sections were placed in citric acid antigen retrieval solution, and after 15 min of high-heat boiling in a microwave oven, the power was turned off and the solution was naturally cooled to room temperature; The endogenous peroxidase blocker was added dropwise on the tissue, incubated at room temperature for 15 minutes, washed with double distilled water; normal sheep serum was added dropwise for blocking, incubated at room temperature for 20 minutes; the section was added dropwise with PD-L1 antibody or IgG antibody (rabbit anti-mouse, 1:200), incubated at 4°C overnight, removed and incubated at room temperature for 30 minutes, washed with PBS for 5 minutes x 3 times, and the incubation of the first antibody was completed; the secondary antibody (goat anti-rabbit, 1:200) was added dropwise, incubated at 37°C for 30 minutes, washed with PBS for 5 minutes x 3 times, and the incubation of the secondary antibody was completed; the working solution of DAB color developing solution was added dropwise, incubated for 10 seconds and then washed with water; May' s hematoxylin was added dropwise, incubated at room temperature for 2 minutes; hydrochloric acid alcohol differentiation, alkaline solution blueing, and water washing were performed; the section was sequentially placed in ethanol from high to low for several seconds (ethanol gradient: 80%, 90%, 95%), anhydrous ethanol for 10 minutes, then immersed in xylene for 10 minutes, 1 drop of neutral resin was added for mounting, and the results were observed under a microscope and photographed.

[0076] The results of immunofluorescence staining of tumor tissue sections are shown in Figure 23 The results show that the tumor tissue in the constructed HCC78 tumor-bearing mouse model highly expresses PD-L1, and is negatively expressed in IgG tumor staining.

[0077] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A compound targeting PD-L1, characterized in that, The targeting PD-L1 compound has the structure shown in the following formula (I) or formula (II) or stereoisomers, tautomers, pharmaceutically acceptable salts, hydrates thereof: Formula (I): ； Formula (II): 。 2. An intermediate for the compound targeting PD-L1 according to claim 1, characterized in that, The intermediate of the compound shown in formula (II) is shown in the following formula (IV); Formula (IV): ； In the formula (IV), the structure of -NHBoc is .

3. The method of producing a compound targeting PD-Ll according to claim 1, characterized by, Comprising: After removing the Boc protection of the compound shown in the following formula (III), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain the targeting PD-L1 compound shown in formula (I); After removing the Boc protection of the compound shown in the following formula (IV), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain the targeting PD-L1 compound shown in formula (II); Formula (III): ； Formula (IV): 。 4. The preparation method of the targeting PD-L1 compound according to claim 3, characterized in that, The molar ratio of the compound shown in formula (III) or formula (IV), tetraazacyclododecane tetraacetic acid-succinimidyl ester and N,N-diisopropylethylamine is (23-27):(35-40):(135-160).

5. The method for preparing the PD-L1-targeting compound according to claim 3, characterized in that, The preparation method of the compound shown in formula (IV) comprises: After removing the Boc protection of the compound shown in formula (III), tert-butyloxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzenetriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain a first-order condensation intermediate; After removing the Boc protection of the first-order condensation intermediate, tert-butyloxycarbonyl 6-aminohexanoic acid, 2-(7-azobenzenetriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine are added to carry out condensation reaction to obtain the compound shown in formula (IV).

6. A radiotracer characterized in that, The targeting PD-L1 compound of claim 1 or the targeting PD-L1 compound prepared by the preparation method of any one of claims 3-5 and a radionuclide.

7. The radiotracer of claim 6, wherein, The structure formula of the radioactive tracer is shown in the following formula (V) or formula (VI): Formula (V): ； Formula (VI): 。 8. The radiotracer of claim 6, wherein, the radionuclide is selected from the group consisting of 18 F, 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb 、227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd and 166 Dy.

9. A method of preparing a radiotracer according to any one of claims 6-8, characterized in that, Comprising: Mixing the radionuclide with the targeting PD-L1 compound to obtain a radioactive tracer.

10. The use of the radioactive tracer of any one of claims 6-8 or the radioactive tracer prepared by the preparation method of claim 9 in the preparation of a preparation for detecting the expression level of PD-L1 in a tumor or in the preparation of a preparation for diagnosing a tumor with high expression of PD-L1.

Images